| Object:1.Pretreatment of SD rat bone marrow mesenchymal stem cells(BMSCs)with different concentration gradients of stromal cell derived factor-1(SDF-1).To explore the effects of SDF-1 on the migration and proliferation of BMSCs in a certain concentration range,so as to obtain the optimal pretreatment concentration and guide the in vivo experiment.2.Using the rat as an experimental animal,the model of the adriamycin nephropathy was modified to establish an animal model similar to human chronic kidney disease.3.BMSCs pretreated with SDF-1 were transplanted into chronic adriamycin nephropathy rats by renal artery,Detecting the renal function indexes,renal tissue pathological changes and the distribution of BMSCs in the kidney to verify the role of SDF-1 in promoting the migration of BMSCs and repairing renal parenchymal cells in vivo.4.To explore the mechanism by which SDF-1 mediates the migration of BMSCs to the injured site and improves the repair of damaged kidney.Methods:1.Selected two Healthy male Sprague-Dawley rats weighing 100g-130g,and used to be bone marrow donor.Rinsing the bone marrow cavity of femur and tibia to obtaining BMSCs,separating and purifying BMSCs by gradient density centrifugation and adherence method.identifying cell phenotype by flow cytometry,and labeling the cells with green fluorescent protein.2.The experiment was divided into 5 groups according to the concentration of SDF1:SDF-10 ng/ml(control group);SDF-1 10 ng/m1;SDF-1 50 ng/ml;SDF-1 100 ng/ml;SDF-1 200 ng/ml.Performing Transwell in vitro chemotaxis experiments to study the effect of SDF-1 on the migration of BMSCs.Performing the MTT assay,and drawning the cell growth curve according to the OD value to exploe the effect of SDF-1 on the proliferation activity of BMSCs.Selecingt the best SDF-1 concentration and co-culturing with BMSCs for 6 hours in advance for pretreatment.Identifiing the osteogenesis and fat-forming ability of BMSCs after SDF-1 pretreatment.3.Detection of renal function and renal pathological changes in rats after transplantation of BMSCsA chronic kidney disease model was established by injecting doxorubicin twice from the tail vein using the male rats weighing 300 g-350 g.On the 4th weekend after the last infusion of doxorubicin,60 rats with successful modeling were randomly divided into 3 groups(model group,BMSC group,SDF-1/BMSC group).The BMSC group was injected with BMSCs not pretreated with SDF-1;the SDF-1/BMSC group was injected with SDF-1 pretreated BMSCs;the model group was not infused with BMCSs,but only with the same amount of medium.All three groups were infused with 1.5 ml of cell suspension by renal artery interventional route,and the number of cells was 1×106.At the same time,20 healthy rats were randomly selected as the control group.Similarly,the same amount of medium was infused through the renal artery pathway when the other groups were transfused with BMSCs.At the same time,20 healthy rats were randomly selected as the control group,Similarly,the same amount of medium was infused through the renal artery pathway when other groups were transfused with BMSCs.Rat BUN,Scr,ALB and 24h urine protein were detected on the day of BMSCs transplantation and 2 weeks after transplantation.HE staining was used to observe the pathological changes of renal tissues,and the distribution of green fluorescence in renal tissues was observed.Detecting the expression levels of some cytokines(VEGF,IL-1?,TNF-?,TGF-?)in rat serum by ELISA.Result:1.The fourth generation of rat BMSCs showed a uniform shape and a long spindle shape.The positive rates of cell surface antigens CD45 and CD11b were 2.2%and 1.9%,and the CD29 and CD44 were as high as 97.1%and 97.9%.Transfection rate with green fluorescent lentivirus is 85%.2.Transwell in vitro chemotaxis assay results:compared with the control group(0 ng/ml),the number of cells chemotaxis increased significantly in each experimental group(10 ng/mL,50 ng/mL,100 ng/mL,200 ng/mL)(P<0.05).When the concentration of SDF-1 was in the range of 10-100 ng/mL,the chemotactic effect of SDF-1 on BMSC was dose-dependent,and the number of chemotactic cells was the highest when the concentration of SDF1 was 100 ng/mL.Compared with the 10 ng/mL group and the 50 ng/mL group,the difference was significant(P<0.05).When the concentration continued to increase to 200 ng/ml,the cell number was not significantly increased compared with the 100 ng/mL group.The results of MTT showed that the OD value of SDF-1 10 ng/mL group,SDF-150 ng/mL group,SDF-1 100 ng/mL group and SDF-1 200 ng/mL group was significantly higher than that of the control group(P<0.05);and between the SDF-110 ng/mL group,the SDF-1 50 ng/mL group,and the SDF-1 100 ng/mL group,there was a statistical difference between each other,which indicating that SDF-1 was concentration-dependent promoting BMSCs cell proliferation(P<0.05).There was no significant difference in OD between SDF-1 100 ng/mL group and SDF-1 200 ng/mL(P>0.05).The experimental results are consistent with the Transwell experimental results.After SDF-1 was co-cultured with BMSCs for pre-treatment,lipid droplets and calcium nodules in BMSCs were observed after differentiation.3.Renal function:Compared with the control group,SDF-1/BMSC group,BMSC group and model group showed significant increase in Scr level(P<0.05),but the difference of BUN was not obvious(P>0.05)on the day of transplantation.After 2 weeks of transplantation,the Scr,urea nitrogen,and 24-hour urinary protein excretion in the SDF-1/BMSC group and the BMSC group were significantly lower(P<0.05),and ALB was significantly increased(P<0.05).Compared with the BMSC group,the SRE-1/BMSC group had a more significant reduction in CRE and 24-hour urine protein,and the ALB increase was more significant(P<0.05).4.Pathological results:After 2 weeks of transplantation,HE staining showed that the glomerular mesangial area of the model group was widened and some of the glomeruli were hypertrophied,Segmental hardened.The renal tubules are progressively dilated,protein tube types,inflammatory cell infiltration and mild fibrosis visible between the tubules.The mesangial cells of the BMSC group proliferated in different degrees.Part of the balloon adhesion,as well as epithelial cell regeneration in individual renal tubules,protein casts in the tubules,and interstitial inflammatory cell infiltration reduced.In the BMSC/SDF-1 group,a small amount of cell proliferation was observed in the glomerular mesangial area,most of the glomerular capillary vasospasm was dilated,the renal tubules were not significantly dilated,the renal interstitial inflamnatory cells were less infiltrated,and the fibrosis was not obvious.5.Distribution of BMSCs in renal tissues:Two weeks after stem cell transplantation,there was almost no green fluorescence distribution in the model group and the control group under confocal fluorescence microscopy;green fluorescence was distributed in the renal tubules but a small amount was distributed in the mesangial area of the BMSC group;in the BMSC/SDF-1 group,Green fluorescence is not only distributed in the renal tubules,but also in the glomeruli.6.Cytokine expression:After 2 weeks of stem cell transplantation,the expression of VEGF of the BMSC group and SDF-1/BMSC group was significantly higher than that of model group(P<0.05),and the SDF-1/BMSC group was higher(P<0.05);In the BMSCs group serum TNF-? decreased(P<0.05),while IL-1?,TGF-?expression levels did not change(P>0.05);in the SDF-1/BMSC group,The expression of IL-1?,TNF and TGF-?1 wereall decreased(both P<0.05),and the level of TNF-? was significantly higher than that of BMSCs group(P<0.05).Conclusion:1.In vitro,SDF-1 can enhance the migration and proliferation of rat BMS Cs,and the number of cells migrating within a certain range is SDF-1 concent ration-dependent.Under the intervention of SDF-1 100ng/mL,BMSCs have the strongest.Chemotaxis and proliferative activity.2.In the chronic adriamycin-induced nephropathy model group,infusion of SDF-1 100 ng/mL pretreated BMSCs by renal artery intervention can significantly increase the number of cells and survival rate of cells targeted to the injured kidney.The conclusions of the experiments in vitro and in vivo are consistent.3.SDF-1 pretreatment can promote cell paracrine effect.For example,inhibiting the accumulation of macrophages,reducing extracellular matrix deposition,increasing neovascularization,delaying renal fibrosis,and improving renal function. |
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